Enzymes immobilized by means of a crosslinking agent, the art to which this invention relates, are widely employed in large quantities and a number of methods have been proposed to immobilize enzymes.
In one method, a carrier is first activated by the crosslinking agent, and then treated with the enzyme, which thus becomes firmly attached to the carrier. Such an activation may include, for example, impregnation of the carrier with a polyamine then treatment with an excess of glutaraldehyde, as is described in U.S. Pat. No. 4,292,199, or in Biotechnology and Bioengineering, 22, pp. 271-287, 1980.
Another activation and crosslinking method suggested to the art involves coating of a carrier with adsorption-promoting insoluble polymer, subsequent adsorption of the enzyme on the polymer, and further, crosslinking in situ, as is described in U.S. Pat. No. 3,705,084.
According to the method described in U.S. Pat. No. 4,069,106, a keratin-containing carrier is activated by reduction of the keratin and the enzyme is crosslinked to the keratin via S-S groups.
U.S. Pat. No. 3,802,909 suggests fracturing glass in the presence of a protein, thus providing freshly produced active sites to bind the protein. U.S. Pat. No. 3,519,538 suggests treating glass with a sequence of chemicals to produce the desired active sites. A related method for producing an immobilized enzyme is described in Biochemical and Biophysical Research Communications Vol. 36, pp. 235-242, 1969: The enzyme is adsorbed on colloidal silica, without previous activation, and then crosslinked with glutaraldehyde.
All the above mentioned methods are characterized by an attachment of a thin layer of enzyme molecules to the active sites on a carrier, and thus the amount of enzyme so immobilized is limited by the number of active sites available.
A different approach altogether, an approach to which this invention relates directly, is to place a thick enzyme containing layer on the surface of a carrier, whereby only a small fraction of the enzyme molecules is in direct contact with the carrier. This way the amount of enzyme immobilized is no longer in some direct proportion to the surface area of the carrier. The amount of enzyme bound on each carrier granule can be controlled. This approach, however, is more difficult to accomplish, as the relatively large amount of enzyme is difficult to maintain in place during immobilization. Therefore, comparatively little has been published about this approach, despite some obvious advantages for such enzyme forms. One suggestion, as described in Canadian Pat. Nos. 1,011,671 and 1,011,672, is to use an aqueous solution of a water-soluble organic solvent as the crosslinking reaction medium, the organic solvent concentration being kept high enough to keep the enzyme insoluble, yet low enough not to interfere with the crosslinking reaction. A main drawback to this method is its requirement for handling of relatively large amounts or organic solvents accompanied by explosion risks necessitating elaborate safety provisions. Also, enzyme products obtained in this way, heretofore, are believed to be deficient in physical stability and yield, which deficiencies might be due to presence of the organic solvent in the immobilization reaction medium.
Another way to obtain a thick enzyme layer is suggested in U.S. Pat. No. 4,116,771. In a limited volume of water the enzyme is treated with glutaraldehyde and an inert protein before being quickly added to the carrier, then allowed to gel, thereafter the product is granulated and finally dried. The high concentration of the crosslinking agent, which is a consequence of the limited amount of water present is often detrimental. Many enzymes are sensitive to high concentrations of crosslinking agents and reduced product yield results, because some enzyme is inactivated, or rendered inaccessible by extensive crosslinking, or both.
The difficulty facing the art's efforts to generate thick enzyme layers, on a carrier, for example, is that the reactant proportions are out of balance. To obtain low concentrations of crosslinking agent in the reaction medium, relative large quantities of reaction medium are required. However, in aqueous reaction medium, some of the enzyme dissolves before crosslinking is effective, or if already in solution, some enzyme is lost in the reaction medium, which factors suggest minimizing the volume of reaction medium. However, avoiding those losses by decreasing the volume of reaction medium would necessitate increasing concentration of the crosslinking agent and this generates the other equally serious enzyme losses already alluded to.
The approach adopted in practice of this invention is to prevent any essential part of the solid or dissolved enzyme in a solvent-free aqueous reaction medium from dissolving in or mixing with the aqueous medium, whereby the volume of reaction medium becomes less important, and low concentrations of the crosslinking reagent therein may be employed. The solubility of enzyme in the aqueous reaction medium is hindered insofar as is reasonably possible.
The object of this invention is to provide a novel enzyme immobilization process, one easy to conduct, and productive of immobilized enzymes with good physical stability.
A further object of this invention is to provide a novel enzyme immobilization process with high recovery of enzyme activity in immobilized form.